CA2553710A1 - Abca1 stabilizer - Google Patents

Abstract

It is intended to provide an ABCA1 stabilizer focusing on the HDL neogenesis system which is pharmacologically useful as a preventive/remedy for low HDL
cholesterolemia. Namely, an ABCA1 stabilizer, which comprises a bisphenol compound selected from among probucol spiroquinone, probucol diphenoquinone and probucol bisphenol as the active ingredient, enables the continuous and stable express of ABCA1 by a mechanism completely different from those in the conventional art, thereby increasing blood HDL level. Therefore, it is useful as a preventive/remedy for low HDL cholesterolemia, arteriosclerosis, etc.

Description

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FIELD OF THE INVENTION
The present invention relates to an AT'~-binding cassette transport 1 (ABCA1) stabilizer. The ABCA1 stabilizer comprises, as an effective ingredient, a bisphenol--type compound selected from probucol spiroquinone ( chem~.ca~, name : 2, 9, 9, 11-tetrakis ( l, 1--dimethylethyl ) -14 . 14-dimethyl-13,15-dithiadispiro[5Ø5.3~pentadeca-1,~1,8,31-tetraene-3,1p-dione), probucol diphenoquinone (chemical name: 3,5,3',5'-tetra-t-butyl-q,4'-diphenoquinone), and probucol bisphenol (chemical name: 4,4'-dihydroxy-3,5,3',5'-tetra-'~-butyldiphonyl). These b~.sphenol compounds are metabolites of probucol. The present invention further relates to a prophylactic/therapeutic agent comprising at least one PsBGFsI stabxlxzex. xhe prophylactic/therapeutic agent is used for treating various diseases caused by a decrease in the ABCA1 expression, such as low-HDL (high-density lipoprotein) cholesterolemia and arteriosclerosis. Here, the A'~p i.s an abbreviation for adenosine 5'-triphosphate that is involved in energy metabolism in vivo and plays an important role in acquisition and utilization of energy.
B,PsCkCGRb'UND OE'' THE TNVENTION
~a Fxobucol is a compound having the chemical name 4, 4' - ( isopxopy7.~.denedithio ) bis ( 2, 6-di--t-butylphenol ) . Main functions of probucol are said to promote catabo~.~,sm of cholesterol and its excretion into bile. Furthermore, probucol raises the catabolism rate of low-densa.ty yipoprotein (LDL)-cholesterol and reduces the serum total cholesterol level. Consequently, probucol is widely used as an agent for s.mpxoving lipid metabolism i,n hyperlipidemia patients (including familial hypercholesterolemia and xanthoma patients).
However, probucol also has a clinically disadvantageous effect, i.e_, reduces the cholesterol level in the IiD~ traction (hereinafter sometimes referred to as "YIDL-cholesterol"), unlike other lipid~lowering agents which have the reaction properties in LDL such as statins ar fibrates (for example, refer to Non-Patent Documents 1 and 2). As the statin lipid-lowering agents, pravastatin and simvastatin known as HMG-CoA xeductase inhibitors axe known. As the f~.brate lipid---lowering agents, fenofibrate and bezafibrate are known. It is thought that .his reduction in HDL--cholesterol is due to functional ~.5 inhibition of ABCA~. (for example, refer to Non-Patent Documents 3, 4, and 5).
It is known that probucol spiraquinone, probucoJ.
diphenoquinone, and probucol bisphenol according to the present invention are produced as metabolites when probucol is orally administered to a mammal (for example, prefer to Non-Patent Document 6).
Some pharmacological activities of probucol spiroquinone, probucol diphenoquinone, and probucol bisphenal (hereinafter sometimes preferred to as "bisphenol-type compounds", or collectively the "bisphenol-type compound") are known at present. For example, zt ~.s disclosed that p7CObucoJ, ba_sphenol has antioxidant properties and is used in combination with probucol as a lipoprotein oxidation inhibitor (for example, refer to Patent Document 1). In addition, it is known that the bisphenoJ.-type compounds incorporate cholesterol into cElls (for example, refer to Non-Patent Document 7). Howe'Ver, iri these psios findings, functions of the bisphenol-type compounds on ABCA1 and HDL are not disclosed at all.

HDL is a lipid/protein complex particle produced by the action of helix-like apolipaprateins such as apoprotein A-I (hereinafter sometimes referred to as "apo,Flx") maznly synthesized in and secreted from liver cells and small-intestine epithelial cells and the ABCA1 protein present iri dell membranes. Tmmediately after secretiall, HDL is ~axmed as a discoidal particle composed of major eanstituents, apoAI and phospholipid, end called nascent HbL. This nascent HDL receives, in blood, free chol.estexo~, from cell membranes of peripheral cells or surfaces of other lipoproteins, and foams mature spherical ~.0 HDL whi.~e holding, at its hydrophobic center, cholesterol ester converted from the cholesterol by the action of LCAT
(lecithan cholesterol acyltre.ns,ferase).
In the above-mentioned process, HDL plays a major role in extremely important physialog,ical function in terms ~.5 of lipid metabolism aa~.J.ed "cholesterol reverse-transport system" which takes, in blood, e~tcessive cholesterol out of peripheral tissues and transports it to the li'v'er. fhe cholesterol reverse-transport system is considered to work for removing cholesterol accumulated in blt~od vessel. wall ~0 cells and to cause a prophylactic action an arteriosclErosis.
With respect to a relationship between blood levels of HDL cholesterol and arteriosclerosis, many epidemiological studies have been conducted. As a result, 25 it has been recently revealed a fact that lower HDL
cholesterol leictels xesu~.t in a higher incidence of arteriosclerosis. The improvement of low HDL-cholesterolemia is a more important and nwcrel technology as prophylactic/therapeutic treatment of arteriosclerosis, 30 compared to a therapy using the statins or fibrates wideJ.y used at present for reducing LDL.
At present, the blood level of HDL is determined by referring to the level of HDL-cholesterol. xn general, when the blood HDL chalestexol. level of a subject is lower 35 than 40 mg/dl, the subject is diagnosed as "low-HDL
cholesterolemia".
Low-HDL cholesterolemia is found as a, risk factor at a high incidence in not only arteriosclerosis but aJ.so in various disorders such as hyperlipidemia, myocardial xnfaxctian, cezebral infarction, Cexebxa~. apoplexy, obesity, diabetes mellitus, and nerve disorders caused by diabetes mellitus. Low-HDL cholesterolemia is also caused by vax~.ous genetic diseases incJ.udirig Tangier disease.
However, a useful prophylactic/therapeutic agent that acts on HDL itself has been desired to be developed. Such a pxophylactia/thexapeutiC agent for treatment o~ J.ow-HDL
cholesterolemia has not been found yet.
xri order td treat low--HDL cholesterolemia, a number of trials for increasing HDL have been conducted.
As a result of such trials, phaxmacoJ.ogxcal, effects of ABCAI have been identified.
ABCA1 is a protein mainly present in cell.
membranes of vaxi.ous organs such as the liver, small intestine, placenta, and adrenal. gland, and belongs to the ABC protein family that is considered to be invoJ.wed in membrane transport of various substances such as lipids, amino acids, vitamins, and saccharides (for example, refer to Non-Patent Document $).
A recent finding revealed that ABCA1 was a protein indispensable fox' a reaction generating HDL from lipids in cells and a rate-limiting factor of HDL
production. In addition, it was revealed that the HDL
formation by ABCA~, is a main removing pathway of cellular cholesterol.
for example, irt patients with fangiex d~.sease whose ABCA1 gene has a mutation and who are deficient in expressing 1~BC~a.~., p~.asma HDL almost disappears (for example, refer to Non-Patent Documents 9, 10, and ~.~.). In addition, it was found that incorporation of A8CA1 gene accelerates a HDL-generating reaction (for example, rE~erred to Nan-patent l~acuments 7.2 and ~.3 ) . Several trials are now in progress to elevate or regulate HDL cholesterol levels by increasing the ABCAI expression level in vivo with genetic engineering technology.
Por example, for increasing cholesterol efflux and HDL levels, the expression level and activ~.ty of RBCAl are elevated by direct gene transfer of an AHCA1-coding gene into a host ceJ.~. (fox example, refer 'to Patent Documents 2 and 3). The expression and activity of ABCAl are increased by using a certain substance to facilitate the transcription and translation of the ABCA1 gene fox controlling the levels of HDL cholesterol and triglyceride (for example, refer to Qatent Document 4). fuxthex7more, far controlling the cholesterol efflux to the outside of cells, the expxesszon of ,~DC,bLI. is increased by activating peroxisorne praliferator activated receptor-a (PPAR-a,) or peraxisome proliferator activated receptorJs (PPAR-$) having 1.5 various activities as an intranuclear receptor (for example, refer to Patent Document 5).
However, in the above-mentioned known technologies focused on ABCA1 and HDL, a genetic engineering technology or a method for actavating an intranuclear receptor is used. Therefore, there are disadvantages such that the technology for a genetic therapy ~.s xmmatuxe and that a r~.sk of unexpected s~.de effects caused by activating an unknown gene is not ignorable. Thus, the use as a drug has not been accomplished yet.
Patent Document 1~ International Publication WO 02/04031 CPatent Document 2~ znternational Publication w0 00/78971 Patent Document 3~ International Publication WO 00/78972 Patent Document 4~ International Publication WO 01/15676 (Patent Document 5~ Japanese Unexamined Patent Application Publa.cation No. 2003--12557.
(Non-Patent Document l~ CTRCULATZOI~T, (T.7S) , 79, 1989, 16--2B
INon-Patent Document 2~ fOURNAL of CARDIOVASCULAR
3S PHARMACOLOGY, (US), 30, 1997, 7$4-7$9 CNon,Patent Document 3~ $IOCHEMTSTRY, (US) , 35 00) , 3996, 1.3021-13020 _(_ [Non-Patent Document 4] BIOCHIMICA et BIOPHYSYCA ACTA, (Netherlands), 1983, 2000, [Non-Patent Document 5~ Arteriosclerosis, thrombosis, and vascular biology, ), 1, 2401, 394--400 {US 2 LNon-Patent Document 5] ANALXxxC.~ CFiEMxS'IFtY SYMPOSxA

SE~CIES, (r~S) , 7, , -3$
19$1 35 lNon-Patent Document 7] LZPZDS, (US), 29(7.2), 1994, $19-Non-Patent Document 8~ ANNUAL REVIEW o CELL BIOLOGY, {US), 8, 1992, 6?-113 Non-Patent Document 91 NATURE GENETICS, {US), 22, 1999, (Non-Patent Document 10~NATURE GENETICS, {US), 22, 1999, 34?351 7.5[Non--Qatent Document11]NATURE GENETICS, {US), 22, 1999, [Non-Patent Document 7.2]THE JOURNAL of CLINICAL

2NVESTIGATION, (US), 104,1999, R25-R31 [Nari-Patent L7oaurnent7.3]THE FASEB ,TOURNAL, {US) , 15, 2001, SUMMARY OF THE ZNVENTTON
~5 AS deSGx~.bed above, low-HDL cholesterolemia is often observed in hyperlipidemia, obesity, and diabetes mellitus and is a serious xa.sk factor of arterioscleroses such as myocardial infarction, cerebral in~axction, and cerebral apvplexx. xhe pxesent invention provides a prophylactic/therapeutic agent for various diseases such as arteriosclerosis based on mechanisms wherein ABCA1 is stabilized with a specific bisphenol-type compound, thereby resulting in elevating ABCIaI levels followed by an increase in production of HDL.
The present inventors have studied for various materials in order to overcome the above-meni~.aned ..7_ d,~sadvantages in the conventional. method and to find an agent affecting an HDL-generating mechanism and being useful as a prophylac~tic/therapeutic agent for low-HDL
cholesterolemia. As a result, the inventors have found a.
prophy7.actic/thexapeutic agent for low-HDL cholesterolemia and filed a patent app7.ication already publ~.shed as WQ
03/033023 Al. The agent contains an effective amount of a cysteine-pz-otease inhibitor which suppresses or inhibits the degradation of ABCA1, thereby resulting in elevated AHCA1 levels.
The present inventors have further conducted extensive studies and succeeded in finding that the aforementioned bispheno7.-type compounds have actions of ~.5 suppressing or inhibiting the degradation of ABCIa.J, to cause the continuous and stable expression of ABCAl, thereby leading to elevated Fit?h levels. Thus, the present invention has been provided. The HDL revel-elevat~.ng action o~ the bisphenol-type compounds is quite contrary to the function of probucol which has been hitherto known to decrease the HDL level relying an inhibition of the AB~A~.
function. Hence, the present invention is not predictable at all from the above-mentioned known technologies. Thus, the present invention prov~.des an agent which overcomes the defect of probucol.
The present invention provides an ABCla,~.
stabilizer comprising an effective amount of a bisphenol-'~ype compound selected from probucol spiroquinone, probucol diphenoquinone, probucol bi.sphenol, and salts thereof (hereinafter sometimes simply preferred to as "the ba.sphenol-type compounds" or "said bisphenol-type compounds"); a prophylactic/thexapeutic agent for low-HDL
cholesterolemxa, which comprises an effective amount of said A8CA7. stabilizer; a prophylactic/therapeutic agent for arteriosclerosis, which comprises an effective amount of said ABCAI stab~.7.izera and a drug compris~.ng an effective _$_ amount of at least one member selected Pram the group consisting o~ said ABCAI stabilizer, said pxophylaGtic/ther~peutic agent for low-HDL cholesterolemia, and said prophylactic/therapeutic agent for arteriosclerosis, in combination with an effective amount of at least one drug selected from the group consisting of antidiabetes drugs, therapeutic drugs for complications of diabetes, antiobesity drugs, antihypertensive drugs, antihyperlipxdernxc drugs, diuretics, antithrombotic drugs, and anti-Alzheimer drugs.
The present invention further provides an agent fox 1) elevatXng the in viya level of A8CA1 and accelerating in vivo HDL-generating reaction, 2) elevating the blood level of HDL, 3) increasing the activity of the blood cholestexdl reverse transport that removes cholesterol from peripheral tissues, and/or 4) suppressing or inhibiting the degradation of ABCAI and accelerating the H~L-generating reaction in vxvo, whXch comprises an effective amount of said bisphenol-type compounds. xhe present invention further provides a prophylactic/therapeutic agent for low-HDL cholesterolemia, which comprises an affective amount of said agent according to any of the above 1) to 4); a prophylactic/therapeutic agent for arteriosclerosis, which comprises an effective amount of said agent according to any of the above 1} to 4)~ and a drug cQmpriszng an effective amount of at least one agent according to any of the above 1) to 4), in combinative with an effective amount of at least one drug selected from the group consisting of antidxabetes drugs, therapeutic drugs for complications of diabetes, antiobesity drugs, antihypertensive drugs, antihyperlipidemic drugs, diuretics, arit~thxombOtic drugs, and anti-Alzheimer drugs.
Tha present invention further provides A) a method for a) stabilizing ABCA1, b) increasing the ABCAI

level and accelerating the HDL-generating reaction in vivo, c) increasing the blood level of HDL, d) increasing activity of the blood cholesterol reverse-transport system which removes cholesterol from peripheral tzssues, and/or e) suppressing the degradation of ABCA1 and accelexatxng the HDL-generating reaction in vivo, which comprises administering an effective dose of the bisphenol-type compounds to a subject (or patient). The present invention further provides B) a method fox pxophylaatic/therapeutic treatment o~ low-HDL cholesterolemia which comprises administering an effective dose of any one of the above-mentioned agents to a subject; C} a method fox prophylactic/thexapeutxc treatment of arteriosclerosis, which comprises administering an effective dose of any one of the above-mentioned agents to a subject (os patient);
and D) a method for prophylactic/therapeutic treatment of a disease, which comprises admznistexing an effective dose of any one of the above-mentioned agents, in combination with an effective dose of at least one drug selected from the group consisting of antidiabetes drugs, therapeutic drugs for complications of diabetes, antiobesity drugs, antihypestensive drugs, antihyperlipidemic drugs, diuretics, antithrombotic drugs, and anti-Alzheimer drugs.
ADVANTAGEOUS PROFILES O~ THE INVENTION
The ABCA1 stabilizer according to the present invention, whzch contains, as an effective ingrediEnt, a bisphenol-type compound selected from probucol spiroquinone, probucol diphenoquinone, probucol bisphenol, and salts thereof, facilitates, free of requiring genetic engineering techniques, continuous and stable T~SCA1 expression with a mechanism completely different from the conventional 3S technologies. The ABC~I stabili~ex exextS the effiCaCy of ameliorating various diseases, such as low-HDL
cholesterolemia, which arise due to a decrease in the ABCAI

expxess~.on. zn add~.tion, since the stabilizer utilizes a metabolite of probucol which has been used as a drug, it is advant~egeous as a highly safe pharmaceutical drug. The present a.nventa.on further provides an agent for increasing the ABCAl level and accelerating the HDL-generating reaction in vivo, increasing the blood level of HDL, enhancing the activity o~ blood cholesterol reverse-tsansport system which tapes up cholesterol fxottt pexxphexaJ.
t7.ssues, arid/ox suppxessi.ng the degradation of ABCA1 and accelerating the HDL-generating reaction in yiyo, which cori~taxns at J.east one of the bisphenol compounds as an effective ingredient. The present invention further provides a method for prophylaoti.c/thexapeutic treatment of various diseases or abnormal conditions (specifically, being associated with a decrease in the HDL blood J.eveJ.) which comprises administering at least one of the bisphenol-type compounds.
The above objects and other objects, features, advantages, and aspects of the present invention are readily apparent to those skilled in the art from the following disclosures. Tt should be ursdexstood, however, that the description of the specification including the following best modes of carrying out the invention, specific examples, era- is xlJ.ustrating preferred embodiments of the present invention and given only for .z7.lustxative purposes. Tt will become apparent to the skilled in the art that a great number of variations and/or alterations (or modificationsy of this invention may be made based on knowledge from the disclosure in the following parts and other parts of the speaifzcat~.on without departing from the spirit and scope thereof as disclosed hezein. All of the patent publications and reference documents cited herein for il~.ustrative purposes are hereby incorporated by reference ~.nta the present disclosure.

B~s~ mtSD~s aF cARRxzNG c~Ux ~H~ xNVELaTIaN
The present invention provides:

2) an ABCA1 stabilizer comprising an effective amount of at .east one bisphenol-type compound selected from probucol spiroquinane, probucol diphenoquinone, pxobucol bisphenol, arid saJ.ts thereof;
2) a prophylaetic/therapeutic agent for low-HDL
cholestero~.emia which comprises at least one A8CA1 stabixi,zex according to the above 1);

3) a prophylactic/therapeutic agent for arteriosclerosis which campx~.ses at J.east one ABCA7.
stabilizer according to the above 1): and 4) a drug comprising at least one member selected 2.5 from the group consisting of ABCA1 stabilizers, prophy7,actic/therapeutic agents for low~HD~, cholesfierolemia, and pxophyJ.actic/therapeutic agents for arteriosclerosis, according to any of the above 1) to ~) , a.n comb~.ria.t~.on with a'~ least one drug seJ.ected from the group consisting of antidiabetes drugs, therapeutic drugs fox complications of diabetes, antiobesity drugs, antihyperteriszve drugs, antihyperlipidemic drugs, diuretics, antithrombotic drugs, and anti-Alzheimer drugs.
~5 The present invention further p7~av~.des:

5) an agent for increasing the in vivo level of ABCA1 and accelerating the in yiyo HD~-generating reaction which comprises an effective amount of at .east one bisphenol-type compound selected from pxobucol. spiroquinone, probucol diphenoquinone, probucol bisphenol, and salts thereog;
5) a:n agent tar increasing the blood level of HDL
which comprises an effective amount of said bisphenol compound:
7) an agent for increasing the activity of cholesterol reverseltransport that takes up, in blood, cholesterol from peripheral tissues, which comprises an effective amount of said bisphEnol-type compounds:
8) an agent for suppressing ox a.nhibiting the in va.vo degradation of A~CA~. and accelerating the in vivo HDL-generating reaction which comprises an effective amount of said bisphenol--type compounds;
9} a pxophylactic/therapeutic agent for low-HDZ
cholesterolemia which comprises an effective amount of at least one member selected from agents according to the above 5) to 8);
1D 10) a prophylactic/therapeutic agent fox axtexa.asalerasis which comprises an effective amount of at least one member seJ.ected from agents according to the above 5) to B); and 11) a dxug compri.sa.z~g an effective amount of at i5 least one member selected from agents according to the above 5 } to 10 ) , a.n combinati.on w3.th at least one drug selected from the group consisting of antidiabetes drugs, therapeutic drugs for complications of diabetes, antivbesity drugs, antihypertensive drugs, 20 antihyperlipidemic drugs, diuretics, antithrombotic drugs, and anti-Alzheimer drugs.
The present invention further provides:
a) a method for stabilizing AHCA1 which comprises 25 administering an effective dose of at. least one bisphenol-type compound selected from probucol spiroquinone, probucol diphenoquinone, probucol bisphenol, and salts thereof to a subject;
b) a method for increasa.ng the in vivo level of 30 ABCA~, and acceJ,erating HDL-generation in vivo which comprises administering an effective dose of said bisphenol-type compounds to a subject:
c} a method for increasing the blood level of HD~
which compr~.ses administering an effective dose of said 35 bisphenvl-type compounds to a subject:
d) a method for increasing the activity of cholesterol reverse-transport that takes up, in blood, cholesterol from peripheral tissues, which comprises administering an effective dose Qf said bisphenol-type compounds to a subject;
e) a method for supprESSing or inhibiting the deg~:adat~.on of A~CA1 and accelerating in vivo HDL-generation which comprises administering an effectXve dose of sa~.d bxsphenol-type compounds to a subject:
t} a method for prophylactic/thexapeutic treatment of ~.ow-HDh cholesterolemia which comprises adrninistexing an effect~,~re dose of at least vne agent according to any of the above 1) and 5) to 8) to a subject;
g) a method for grophylaatic/ther~peutic treatment of arteriosclerosis which comprises administering an effective dose of ~.t leas' orie agent acc4xding to any of the above 1) and 5) to 8} to a subject: and h) a method for prophy~.actic/therapeutic treatment of a disease which comprises administering an effective dose of at least one agent according to any of the above 1) to 3) and 5) to 10), in combination with an effect~.ve dose of at least one drug selected from the group consisting of antidiabetes drugs, therapeutic drugs for compJ.xcatxons of diabetes, antiobesity drugs, antihypertensive drugs, antihyperlipidemic drugs, diuret~,cs, antithromboti.c dxugs, and anti-Alzheimer drugs.
~5 The preseri'~ ~.nver~tion pxovides A) a pharmaceutical drug containing, as an effective compvnen'~, a bisphenol--type compound selected from probucol spiroquinone, probucol diphenoquinone, probucol bisphenol, and salts thereof: and 8) a pharmaceutical composition containing a pharmaceutically effective dd$e of a bisphenol-type compound selected fxom probucol spiroquinone, probucol diphenoquinone, probucol bisphenol, and salts thereof, in admixture with a phaxmaceuta.caZ7.y acceptable e~tcipient.
The above-mentioned probucal sp~.xoqu~.none, probucol diphenoquinone, and probucol bisphenol can be chem~.ca~.~.y synthesized by techniques, for example, disclosed in the Examples herein below. These compounds can be also isolated and purified by biochemical techniques and chemical techniques, which are generally used, from in vivo metabolites produced when probucol is adma.nzstexed to a mammal.
Among the aforementioned bispheno~.--type compourids, when they are capable of forming salts, preferable salts thereof encompass pharmaceutically acceptable salts, including, for example, salts with inorganic or organic bases; salts with neutxal, bas~.c, ox aci.d~.c amirio acids;
etc. Preferable examples of the inorganic bases include alkaline metals such as sodium and potassium: alkali earth metals such as calcium and magnesium; alurni.ttum: ammonium:
etc. Preferable examples of the organic bases include trimethylamine, triethylamine, pyridine, pxcvlxne, ethanol.arna.ne, da.ethanolamine, triethanolamine, dicyclohexylamine, N,N-dibenzylethylenediamine, etc.
Preferable examples of the neutral amino acids include glycine, valine, leucine, etc. Preferable examples of tha basic amino acids include arginine, lysine, orn~.thine,~eta.
preferab~.e examples ofi the acidic amino acids include aspartic acid, glutamic acid, etc.
The ASCAI. stabilizer is an agent Car drug that provides the cont7.riuous and stable expression of ABGA1 mainly pxesent in cell membranes of various organs such as liver, small intestine, placent~3, and adrenal gland. The stabilization of ABCA1 may refer to a state in which ~J-3CA1 is being continuously and stably expressed. Examp~.es of such a state may include, when ABCAI levels are compared between in the presence and absence of the ABCA1 stabilizer, a state in which ABCA1 is more continuously and stably pxesent in cells (in particular, in cell membranes), a state in which ABCAI is present ~.n cells (in particular, in cell membranes) so as to further accelerate the YiDL-generating reaction, and a state in which ABCA1 is present.
in cells (in particular, in cell membranes) so as to further suppress ox l.nh,i.bzt the degradation of RBCRJ., under the effect of the ABCA1 stabilizer.
The prophylactic/therapeutic agent for low-HDL
cholesterolemia is a drug that prevents, relieves or cures low-HDL cholesterolemia. Low-HDL cholesterolemia is observed in diseases such as, in particular, 2D arteriosclerosis, hyperlipidemia, myocardial infarction, cerebral infarction, cerebral apoplexy, obesity, diabetes melJ.itus, nerve disorders caused by diabetes rnelLitus, thyroid dysfunction, hepatocirrhosis, myeloma, chronic renal failure, and chronic ~.nflarnrnatory bowel disease 1,5 (examp7.es: Crohn~s disease, chronic ulcerative colitis).
The prophylactic/therapeutic agent for low-HDT
cholesterolernia comprising the ABCAl stabilizer according to the present xn,srent~.on can be used for prophylactic/therapeutic treatment of any of the above-20 mentioned diseases excepting genetic diseases, such as Tangier disease, in which ABCA~. ~.s not normally synthesized i.n viva.
The pxophy~.actic/therapeutic agent for ?5 arteri.oscl.erosis is a drug that prevents, relieves or auras arteriosclerosis- fhe R.8CA1 stabilizer according to the present invention potEntly act in acceJ.exating HDL-generation to increase blood HDL levels (or increase plasma HDL levels or serum HDL le~crels) as it will be clear from 30 Rssay Examples herein below. The blood HDL plays ari lmpOrtant role in the cholestezo~. reverse-transport system which prophylactically acts an arteriosclerosis. The drug of the present invention is also useful as a prophylactic/therapeutic agent far arteriosclerosis via the 35 aC'tian of increasing the blood HDL level.
In other words, in low-HDL chalesterolemia, the cholesterol, reverse-transport system cannot suffic~.ent,ly work due to a decrease in the HDL level. Consequently, ahol.estexol accumulated ~.rs vascula:: walls is nat readily transferred to the outside of the blood vessel, thereby resulting in acceleration of arteriosclerosis. Such a condition can be ameliorated with the prophylactic/therapeutic agent for low-HDL cholesterolemia according to the present invention. Thus, the agent can be prophylactically and therapeutically applied to arteriosclerosis. Similarly, the bisphenol-type compounds i0 are useful fax' increasing the A8CA1 level and aaceleratXng the HDL-generating reaction in vivo; increasing the blood level o~ fIDL; increasing the cholesterol reverse-transport system activity in blood, which takes up cholesterol from peripheral tissues: and/or suppressing ar inhibxtxz~c~ the 7.5 degtadati.on of ~.1BCA1 arid accelerating the HDh-generating reaction _in vivo. Therefore, the bisphenol compound can be .
used for prophylactic/therapeutic treatment of low-HDL
cholesterolemia and arteriasclerasis.
20 Tn addition, the ABCA1 stabilizer o~ the present invention is useful for various diseases which are thought to be caused by a decrease in the .ASCA~. expxessxor~, as follows:
Coxonary artery disease (including cardiac 25 ~.ntaretion, angina pectoris, asymptomatic myocardial ischemia, and coronary arteriasclexoszs); atherosclerosis;
carotid arteriosclerosis: cerebrovascular disease (including cexebral apoplexy arrd cerebral infarction);
artexi.osclerosis obliterans; fatty liver; hepatoci.rrhosis;
30 myeloma; diabetes mellitus; diabetic carnplication;
dermatologic disease; xanthomatosis; arthritic disaxdex:
prolifesative disease; peripheral axtex~.a~. obstruction;
ischemic peripheral circulatory failure; obesity:
cerebrotendinous xanthamataszs (Cx~C); chronic renal 35 failure; glomerular nephritis; arterioscleratic nephxXtis;
vascular thickening; vascular thickening aftEr interverlt~.on (including percutaneaus corot7ary pl~,sty, percutaneous coronary revascularization, detention of stmt, coronary endascapy, intxavascuJ.a~ sonicatian, and percutaneous transluminal thrombolytic therapyy: vascular reocclusion and restenosi.s after bypass operation: nephropathy, S nephx~.tis, and panereatitis, which highly relate to hyperlipidemia; hyperlipidemia (including fam~.J.S.a~.
hypex~cl-~o~.estexo~.emia and postprandial hyperlipidemia} ;
chronic infilammatory bowel disease (including Crohn's disease and chronic ulcerative colitis}: a.ntexmittent ~.0 alaud~.cation: deep venous thrombosis; malaria encephalitis:
Alzheimer's disease; and diseases accompanied wzth wound or ateliosis.
The drug containing the active ingredient according to the pxeserit invention is effective for ~.5 prophylactic/therapeutic treatment of various diseases or abnormal condxtzdns which are associated with a decrease in the blood HD~ level, and is obviously useful, for the above-mentioned diseases.
The ABCA1 stabilizer, prophylactic/thexapeutic 20 agent for low-HDZ cholesterolemia, and prophylactic/therapeutic agent for axtexi,osclerosis according to the present ~.nvct'xt~.orr may be administered alone or in combination with other drugs described below, preferably in a form of a drug containing a 25 pharmaceutically acceptable exc~.pzent_ The agent is administered by an oral route or injection. rn addxti.on, the dxug may be zn topical form (such as percutaneous drug or ointment), suppository form (such as xectal suppository and vaginay suppository}, pellet foam, nasal-drop form, 30 inhalant foam (such as a farm using a nebulizer}, or eye-drops form. In any administration route of the drug, a componEnt (hereinafter sometimes zeferred to as "pharmaceutical component") selected from known pharm~.ceutxcaJ. exc~.p~.ents can be optionally used.
35 Specifically, examples of known exCipients axe disclosed in, for example, (1) ryakuhin Tenkabutsu Handobukku (Handbook of Pharmaceutical Excipients), MA RUZEN, x.989, (2} Iyakuhin Tenkabutu Jiten (Encyclopedia of Pharmaceutical Excipients), fist Edition, Yakuji Nippo, 1994, (3) ~yakuhin Tenkabutsu Jiten Tsuiho (Encyclopedia of Fharrnaceut~.ca~. Estcxpxents, Supplement), 1st Edition, Yakuji Nippo, 1995, and (4) S Xakuzaigaku (Pharmacology), Revised 5th Edition, Nankodo, 199. The pharmaceutical component may be optional7.y selected from the known pharmaceutical excipients shown above depending on the adrna.n~.stxat~.on route arid applzcatXon purpose of the drug. Similarly, the agent of the present invention fox ~.ncxeas~.ng the I~BC~. ~.eveJ. and acce~.exatxng the HD~-generating reaction in vivo; increasing the blood level of HD~; increasing activity of the cholesterol reverse-transport system in blood, which removes choJ.estexoJ. from per~.phexal tissues: and/or suppressing the degradation of ABGAl and accelerating the HDTV-generating reaction in vivo may be administered as described above.
For example, when the drug is adm~.n~.stered orally.
any excipient can be used as long as the excipient can constitute an oral drug as a pharmaceutical component and achieve purposes of the present invention. Generally, the exc~.pi.ent is selected from known pharmaceutical components including for example fillers, binders, disintegrants, lubricants, and Gaa.t~.ng agents (including taste masking 2.5 agents). ExampJ.es of the oral drug include tablets (including sublingual tablets and orally d~.sa.ntegrating tablets), capsules (including soft capsules and microcapsules), granules, tine granules, powders, troches, and syrups. The oral drugs include those (examp~,es: xapid-release drugs and sustained-release drugs) in which a known pharmaceutical component is used to control the in vivo release o~ an active component (a..e., an effective infredient), i.e., the in vivo release of probucol spiroquinone, probucol diphenoquiriorie, ox probueol bisphenal.
When the drugs are administered by injection - 1~ -routes, the. excipient used includes any pharmaceutical component which can constitute an aqueous injection or non-aqueous injection. In general, known pharmaceutical components which are used include d~.ssol.vi,ng agents, dXsso~.vxng a~.ds, suspending agents, isotonizing agents, buffering agents, stabilizing agents, preservatives, etc.
In addition, the excipient used may include known pharmaceutical compounds wh~.ch constitute powder injections.
The powder injections are used after being dissolved or suspended when they are administered. Examples of the pharmaceutical components for aqueous injections include distilled water for injection and ste~:ile isotonic salt solutions (containing, for example, monosodium phosphate, disodium phosphate, sodium chJ.oxide, potassium chloride, calcium chloride, magnesium chloride, or a mixture thereof).
Examples of the pharmaceutical components for non-aqueous injections include vegetable oils such as olive oil, sesame oil, cotton oil, and coin o~,l.; propylene glycolr macrogola arid tricapryline. The drugs are prepared by dissolving, suspending, or emulsify~.ng the active component in these pharmaceutical components. Examples of the injections include subcutaneous ~.njecti,ons, intravenous injections, intramuscular injections, intraperitoneal injections, and intKavenaus dips. Provided thus is use of the bisphenol.--type compounds for preparing drugs (phaxznacautical drugs or pharmaceutical compositions) which contain the active ingredient or active component of the present invention and hs~re the above-mentioned activities.
The effective dose of the AECAI stabilizer, prophylactic/therapeutic agent for low-xD~ cholesterolemia, and prophylactic/therapeutic agent for arteriosclerosis according to the present invention varies and is optionally controlled depending on the age and weight of a subject, 3$ symptoms of the low-HDh cholesterolemia ox arteriosclerosis, and the presence or absence of compZications_ In general, the dose is about 0.1 to 3000 mg/day when the agent ~.s administered orally, and the dose is about 0.1 to 1000 mg/day when the agent is administered by injection.
Similarly, the agent of the present invention for increasing the ABCA1 level and accelerating the in vivo HDL-generating reactions increasing the blood level of HDL:
activating oz stimulating the cholesterol reverse transport pathway in blood, which removes cholesterol from peripheral tissues: and/or suppressing the degradation of ABCA1 and accelerating the in vivo HDL-formation may be administered 20 as described above.
The ABGA1 stabilizer, prophylactic/therapeutic agent far low-HDL cholesterolemia, and prophylactic/therapeutic agent for arteriosclerosis according to the present iriven~.ion can be used as a combination with one or more other drugs, which do not adversely affect actions of the inventive agents, in order to increase the efficacy, reduce the dose to be administered, and decrease side-effects. The drug used in the combination may include low molecular-weight compounds, 0 polypeptides, antibodies, vaccines, etc. Such drugs include, fox' example, "antidiabetes drugs", "therapeutic drugs for complications of diabetes", "antiobesity drugs", "antihypertensive drugs", "antihyperlipidemic drugs", "diure'~xcs", "antXthrombotic drugs", "anti--Alzheimer drugs", and others. Similarly, the agent of the present invention for elevating the ABCA1 level and accelerating 'the HDL
formation _in _viva: xncreas~.ng Clue blood level of HDLG
activating the cholesteral reverse--transport pathway in blood, which removes cho~.esterol from peripheral tissues;
and/or inhibiting the degradation of ABC1~1 and accelerating the HDL formation in yiyo may be administered as described above.
The "antidiabe~es drug" includes, for example, insulin secretagogues, biguani.des, insulin, and cc-glucosidase inhibitors. 'the insulin secretagogue includes, for example, tolbutamide, chlorbutamide, ah~.orpropamide, tolazamide, acetohexamide, glyclopyramide, glibenclamide, glielazide, glibuzol, glimepirid, nateglinide, and mitiglinide. The biguanide includes, for example, phenformin, metformin, and buformin. The a-glucosa.dase inhibitor ~.nc~.udes, for exampJ.e, acarbose, eroglibose, miglitol, and emiglitol.
The "therapeutic drug far complica-~ions of diabetes" includes, for example, aldose~reductase inhibitors such as epalrestat, alprostadil, and mexiletine hydrochloride.
The "antzobesity drug" includes, ft~x exarrtpJ.e, lipase inhibitors and appetite suppressants. The lipase inhibitor includes, for example, orlistat. The appetite suppressant includes, for example, dexfenfluramine, fluoxetine, sibutramine, and baiamine.
The "antihypertensive drug" includes, for example, angiotensin-converting enzyme inhibitors, calcium antagonists, and arefxateX~sin xz antagonists . The angiotensin-converting enzyme inhibitor includes, for example, captopril, enalapril, alacepril, delapril, lisinopril, imidapril, beriazepr~.l, c~.lazapril, perindopril, quinapril, temocapril, trandolapril, and manidipine. The calcium antagonist includes, for example, ni,feda.pa.ne, amxoda.pzne, efonidipine, and nicardipine. The anigotensin II antagonist includes, for example, losaru'~an, cardesar'~an ailexatil, vaJ.saxtan, and ixbesartan_ The "ant~.hyperlipidemic drug" includes, for example, HMS-CoA reductase irih~.bxtars and tibrates. The HMG-Cob1 reductase ~.nhibitor includes, for example, statins such as pravastatin, simvastatin, lovastat~.n, atorvastatin, and fl.uvastatin. The fibrates include, for example, bezafibrate, clinofibrate, clofibrate, fenc~fa.bxate, and simfibrate. In addition to the above-merita.oned drugs, the "antihyperZa.pa.demic drug" includes, for example, anion exchange resins (example: cholestyramirse), nicotinic aca.d agents (examples: nicamol and nicexitrol), and ethyl iGOSapentate.
S The "diuretic" includes, for example, thiazide drugs such as cyclopenthiazide, txichloromethiazide, hydrochlorothiazide, hydroflumethiazide, benzylklydrochlorothiazide, penfluthiz~.de, and methyclothiazide. Tn addition to the above-mentioned drugs, the d~.uretic includes isasarb~.de and furosemide_ The "antithrambotic drug" includes, for example, heparin, warfarin, antithrombin agents, thrombalytic agersts, and platelet-aggregation-suppressing agents (anti-plate7.et ~.5 agents).
'the "anti-Alzheimer drug" includes, for example, donepeziZ, rivastigmine, and galantamine.
When the ABGA1 stabilizer, prophylactic/thexapeutic agent for low-HDL cho~.esterolemi.a, or prophylactic/therapeutic ag~:nt for arteriosclerosis according to the present invention is applied in combination with any of the concomitant agents, the dosage form is not specifically limited as long as the agent of 2S the present invention is coadministered with the concomitant agent. Examples of such dosage forms include those for admin~.strating a single dosage un~.t prepared by formulating the agent of the present invention and the concom~.tant agent together; for administrating two kinds of drugs at the same time or at an interval via identical routes, wherea_ri the two kinds of drugs are prepared by separately formulating the agent of the present invention and the conoomitant agent; anct for administrating two kinds of drugs at the same time or at an interval via different ~5 routes, wherein the two kinds of drugs axe prepared by separately fozmulatzrig the agent of the present invention and the concomitant agent.

~23-The present ~.nvent.ion provides a method for prophyLactic/therapeutic treatment of diseases with the bisphenol-type compounds. For example, 'the method far praphylact~.a/thexapeutic treatment of diseases can be performed by administering an effective dose of the bisphenol-type compounds '~o a subject or targe'r.. The inventive method for prophylactic/therapeutic tz~eatment of diseases may be performed while monitoring conditions arid symptoms of a subject. fhe man~.toxzng may be conducted at regular or irregular intervals of time, or may be periodicaJ.ly conducted. Typically, the method is performed while monitoring the blood HDL level. The drugs of the present invention may be adminis'~exed at regular or irregular intervals of time, or may be periodically administered.
The terns "pxaphyZacti.c/therapeutic treatment"
used herein refers to preventive trea'~merit artd/ox curative treatment, i.e., including (1) prophylactic and therapeutic treatments, and (2) praphylaatic txeatrnent or therapeutic treatment.

_ '~G~. ..
Examples, et.c.
Details of the present invention axe described by the followa.ng examples including Synthesis Examples, Assay Examples, and Formulation Examples, but such examples axe provided only for illustrative purposes, and for referential embodiments of the present invention. These examples shou7.d in no way be construed as limiting and restricting the scope of the invention disclosed herein.
~0 It should be understood in the present invention that various embodiments can be made or executed within the spirit, scrape and concept disclosed herein.
All the .Assay Examples and other Examples were performed or can be performed, unless otherwise disclosed herein speCificalLy, according to standardized techniques which are well-known and conventional. to those skilled in the art. Tn Examples herein below, specif~.c processes and treatment conditions were performed, unless specific indication ~.s provided, according to attached protocols with attached reagents when commercially available reagents yr kits were used.
Synthesis Example 1 Synthesis of ptobucol spiroquinone A mixture of probucol (987. mg, 1.9 mmol: Wako Pure Chemical Industries, htd-) and lead oxide (4.04 g) in diahloromethane (20 mZ) was stirred overnight. xhe mixture was ~iltered, evaporated, 'then washed with methanol, and dried to yield a crystal, probucol spiroquindne (912 mg, 935). Chemical ionization mass spectra (Cr-MS) were recorded on a double-focusing magnetic sector mass spectrometer {MS700: JEOL). Elemental analysis was conducted faith a CHN autoanalyzer {vario EL: Elementar}.

mp 156-158°C
1H NMR (CDC13) S: 1.20 (36H, s), 2.01 (6H, s), 6.88 (9H, s) CZ (positive)--MS (m/z) : 515 (M+1) , 473 (M-C~H~) , X141 (M-C3H6S*1) , 409 (1~-C3I~6S,~-rl, dipherioquinone-~1) , 279 (M_ ClqH2bCS+1 ) , 237 ( C14H20CS*1 ) Anal.. ca~.cd_ far C31Hq6p2S2~ C. 72.32: H, 9.01. Found: C, 72.3: H, 9.0 Synthesis Example 2 Synthesis of probucol diphenoquinone A methanol solution (lOb mh) of 2,6-di-t-butylphenol (309 mg, 1.5 mmol) was oxidized under the presence of phthalocyanine-Fe (II) ($53 mg, 1.5 mmol) at a roam temperature far 5 hours aacardi.ng to a method of Tada, et aJ.. (EuZI. Chem_ Sac., 45, 2558-2559, (1972)]. The resultant reaction mixture was stirred until the reaction was completed, and then evaporated. The resulting residue was dissolved in ethanol, filtered, and evaporated to yield a crystal, prabuaal diphenaquinone (34o mg, loo EJ.ectron impact mass spectra (EI-MS) were recorded on a double-focusing mass spectrometer (AX505W: ,~EaL).
mp 223,225°C
1H NMR (CDCI~) &: 1.37 (36H, s), 7.71 (4H, s) ET-MS (m/Z): A08 (M), 393 (M-CH3), 351 (M-GgHg) Synthesis Exarnp~.e 3 Synthesis of probucol bisphenol ~'o a so7.ut~.on of pxobucol diphenoquinone (349 mg, 0.86 mmol), prepared in Synthesis Example 2, ~.n methanol (20 mZ) arid dichJ.oxomethane (20 mT~) was added sodium borohydride (72 mg, 1.8B mmol) under nitrogen atmosphere.
The resultant reaction mixture was starred for 1 hour, evaporated, then washed with water, and dried to yield a crystal, probucol b~.sphenol (244 mg, 70$). Electron impact mass spectra (EZ-MS) were recorded on a double-focusing mass spectrometer (fiX505W: fEOL).
mp 184-186°G
1H NMR (CDClg) 8: 7..49 (36H, s) , a.l$ (213, s) , 7.30 (4H, s) EZ-MS (m/~): 410 (M), 395 (M-CH3) Assay Example 1 Increase in the RBGA1 expression bar probucol spiroquinone, probucoJ. di~henoquinone, and probucol bisphenol in THP-~.
cells Assay method>
'BHP-1 cells (human leukemia cells: American Type Culture Collection) were cultured far ~2 hours in a 10~C
fBS-RPMT1640 medium {Iwaki Glass Co., Ltd.) under 'the presence of PMA {phorbol rnyristate acetate, 3.2 x 7.0 ~ M:
Wako Pure Chemical Industries, Ltd.) to obtain differentiated macrophages. Pxobucol spiroquinone (synthesis Example 1), prabucol diphenoquinone (Synthesa.s Example 2), or probucol bisphenol (Synthesis Example 3) was added to a medium after being incorporated into acetyl--LDL
according to a method of Tsu'ita, et al. [BIOCHEMISTRY, 3S, 13011-1302D {1996)1. The cells were cultured in this medium for 48 hours, and then further cultured for 24 hours in the presence or absence of apoAr. The expression levels of intracellular ABCA1 were assayed according to a method disclosed herein below.
~'he agent-treated cells and non~treated cells were hypotonicaJ.ly disrupted in 5 mM Tris-HCl (pH 8.5), and then centrifuged (650 g for 5 minutes) to precipitate nuclear fractions. The superriata.nt was centrifuged at 7.05,D00 g fox 30 minutes to collect a total membrane fraction. The total membrane was dissolved ~.n a solution containing 0.9 M urea, 0.2$ Triton X-100, and 0.1~
d~.th~.othxe~.toz, followed by addition of IO$ lithium dadecyl _z7_ sulfate solution at a vo~.ume ratio of 1/4. The resultant was subjected to electrophox-etic sEpaxation using 1.~'~ SD5-7~ polyacrylamide gel. The separated proteins were transferred and fixed onto PvDF membrane (Bi.o-Radj, and then subjected to immunoblotting with anti-human A$CA2 rabbi'b antibody (seXf-purified by an ordinary technique) to examine expressed ABCA7. protein levels . The obtai,ried ba,1'ids of 'the protein were read for density and size with Scion Image (image analysis software: Scion] to convert the 1Q results into digit~.z~d forms. The relative ratio of the ABCAZ expression level of the drug~treated cells to that of the non-treated cells was calculated to evaluate the ABCA1-expxessiort acta.vity of the assayed compounds. The results axe shown in Table 1.
CResults~
As shown zn Table 1, it was observed that the i.rltracellular ABCLL1 expression levels of the THF-1 cells treated with probucol sp,zroqua.none, probucal d~.phenoquinone, ox probucol bispheriol were remarkably elevated, as compared with that of the non-treated cells.
Table 1 A~CA1 F,xpression ~eVel Assayed ~omppund {Relative Ratio /a) Contro) (Non-treated) ~ pp Probucol splroquinone ~g~

Probucol diphenoquinone973 Probucol blsphenol 1~g _~$_ Assay Example 2 Acceleration of HDLG-Generating reaction by probucol sp'~xoc(uinone, probueol diphenoauinone, and rirobucol bisphenol Assay method>
The cultured cells obtained ~.n the same manner as in Assay Example 1 were examined for cholesterol and phospholipid taken out into the medium by the apoAx-dependent HDL-generating reaction, according to a method of Arakawa, et al. ~J. LzP~D RES., 41, 152--1962, (2000)]_ The results of the drug-treated cells were compared with that of nc~n-treated cells (control). The resulting data we7~e converted into digitized foams as in Assay Example 1, and the relative ratios of the taken out level in the drug-treated cells to that in the non-treated ceJ.~.s were calculated to evaluate the HDL-generating reaction accelerated by the assayed compounds- The results are shown in Table 2. for comparison, the HiDL-generating reaction accelerated by pxabucol (Wako Pure Chemical Tndustries, Ltd.) was examined in the same manner as aforementioned.
~cRestllts~
As shown in Table 2, it was observed that the levels o~ apoAT-dependently 'taken out HDL cholesterol were about 1.3 to 1.6 times elevated in the THf-1 cells treated with probucol spir4quxnone, probucol diphenoquinone, yr pxobuco~. bisphenol, as compared with that of the non-treated cells. Similarly, the levels of apoAl-dependez~t~.y taken out phospholipid were also about 7..4 to 2.0 times increased in the THP-1 cells treated with probucol spiroquinone, probucol diphenoquinone, or probucol bisphenol, as compared with that of the non-treated cells.
On the other hand, i.n the THP-1 cells treated with probucol, ria apaAl-dependently taken out HDL cholesterol was observed and the level of taken out phospholipid was decreased to _ZC)_ less than 1/3, as comparEd with that of the non-treated celJ.s _ Table 2 Assayed CompoundRelease of HDL ChalestarolRslease of Phospholipid (Relative Ratio elative Ratio %) Control (Non-treated)100 100 Prabuoal spiroquinone148 151 Probucol diphenoquinone159 200 Probuool bisphenol137 144 Probucol 0 3~1 Assay >axamp3.e 3 Increase of blood HDL level by robucvl stoxxottuxnone and ~robucol diphenoquinone (mouse}
<Assay method Probucol spirvguinone (~yt~thesis Example 1: 50 mg/kg or 150 mg/kg} or probucol diphenoguinone (Synthesis Example 2: 50 mg/kg or 150 mg/kg} suspended in a 0.5$
caxboxymethy7.ce~.zuZose solution was orally administered to each group of 3 or 4 mice once a day for 7 days. To a control group, only the 0.5'3 carboxymethylcellulose solution was administered in the same manner. Animals were subjected to laparotomy under ether anesthesia at 3 hours after the last administration, and blood was collected in the presence of heparin sodium from the heart of each mouse.
The blood was centrifuged at 2000 g for 10 minutes to separate plasma. The plasma was applied to electroghores~.s using Paragon Electrophoresis System (Beckman Coulter, Inc.} ~.o separate pJ.asma lipoprotein, followed by lipid staining. The images on the gel after the lipid staining were examined to covert the plasma FIDL level of each mouse into a digitized form for the evaluation.
<Results3 As shown in Table 3, it was observed that the plasma HDL levels in mice administered With 'tewt drugs wexe inareo,sed about 7..2 times for probucol spiroquinone and about 7..1 to 1..8 t~.mes for probucol diphenoquinone, respectively, compared with that in control group.
Table 3 Assayed Compound Plasma HOL
Level (Relative Ratio %) 5D mglkg 150 mglkg Control (Non-treated)100 100 Probucol spiroquinone120 124 Prabucal diphehaquinorie113 180 Assay Example 4 ~ncxease of blood HDL level by probucol spiroquinone,_ robucol di heno uinone, and robucol bis henol (rabbit) <Assay method Microemulsions were prepared by adding probucol spiroquinone (Synthesis Example 1), probucol diphenoquinone (Synthes~.s Example 2), or probucol bisphenol '~o a m~.xtuxe ofi triolein (Sigma Chemical Co.) and phospha.'tidyl choline (Avanti folax Lipids). The microemulsion (0.5 to 10 mL/body) was administered to each group of 4 rabbits via the ear vein once a day for 7 days_ To a control group, a microemulsion containing no test drag was administered in the same rna~x~7ex. Blood was collected in the presence o~
heparin sodium from the eas vein of each rabbit at 3 hours after the last administration. Tha blood was centrifuged at 2000 g fox 7.0 minutes to separate plasma,. The p7.asma was applied to electrophoresis using Paxagon Electrophoresis System (Beckman Coulter, Inc.) to separatE
plasma lipapxote~.n, followed by lipid staining. The images on the gel. after the lipid staining were ex~tmxned to covext the plasma HDI. level of each rabbit into a digitized form for the evaluation. In reference to the content amount of each compound in the micraemulsion, each blood compound level, assumed just after the intravenous adrilina.stxation thereof was set as the dose of each compound.
CResu~.ts>
As shown in Table 4, it was observed that the plasma HDL levels in rabbits administered wa.th test drugs 1.5 were increas~:d about 1.5 to 2.9 t~.mes for probucol spiroquinone, about 1.3 to 1.4 times fox probucol diphenaquinone, and about 1.3 to 1.4 times far probucol bisphenol, respectively, compared with that in control group.
TablE 4 Assayed CompoundPlasma HDL
Level (Reletive Ratio %) Blood Levei:
M

Control (Non-Treated100 Prabuaol spiraquinone1~ 194 0.14 0.57 Probucol 148 130 dfpheno uinane 0.16 (0.31) probucol bisphenol1~ 13' (0.14) 0.

25 Assay Example 5 Toxicity Test Prabuaoz spzroquinone, probucol dipherioqu~.none, and probucol bisphenol were orally administered to mice for 30 one week. As a, xesult, it was verified that na abnormal findings were observed.
As shown above, it was canf~.rmed that the ABCA1 stabilizer of the present invention proUr~.des the continuous and stable expression of AHCAI without using genetic engineering techniques and accelerates the HDL-generating xeact~.on, thereby acting as a prophylactic/therapeutie agent fox ~.ow-HDL cholestervlemia. The active ingfedients, the bisphenol-type compounds, aaaa7:d~.ng to the present invention have activity of elevating blood HD~ levels, and hence are expected to have activity of modifying the cholesterol reverse-transport system to a preferable direction.
7. 5 Eozmulation Example 1 According to the present invention, probucol spi.raqu~.none: 200 mg, lactose: 100 mg, corn starch: 28 mg, and magnesium stearate: 2 mg xhe ingredients of the prescription above were formulated into capsuJ.as according to a known method specified in The Japanese Pharmacopoeia XzV, General Rules for PrEparations.
Formulation Example 2 3C? According to the present invention, probucol spiroquinone (25 mg) was disso~.ved in an aqueous isotonic solution of d~.st~.lled water for injection (~.0 mh) containing an appropriate amount of sodium chloride. The resulting m~.xture was dispensed in each ampu~.e, and then subjected to steriJ.~.zation after sealing to obtain inject~.ons .

INDUSTRIAL APPLICABILITY
The ABCA1 stabilizers each contazna.ng an effective amount of a bisphenol-type compound selected from probucol spiroquinone, probucol diphenoquinone, and probucol bisphenol according to the pzesent ~.nvention give the continuous and stable expression of ABCA1 without using complicated genetic engineering techniques. The ABCA1 stab~.lizers are effective drugs for various diseases such as l.ow~-HDL cholesterolemia and arteriosclerosis caused by a decrease in the ABCA1 expression. In addition, the ABCA1 stabilizers of the present invention utilize a metabolite of probucol which has already been assured to be pharmaceutically safe, and thus are useful as drugs because of their safety. Similarly, the effective ingredierr,ts can be used a~ agents xor ~.ncxeasing the ABCA1 level and accelerating the HDL-generating reac'~xorl i.n vivo~
increasing the blood level of HDL; activa,tinf the b~.ood cholesterol reverse-transport pathway which takes up cholesterol from peripheral tissues; and/or suppressa.ng the degradation of ABCA1 and accelerating in vivo HDL formation, and further can be used for prophylactic/prevent~.ve treatment of ~~rarious diseases or abnormal conditions (specifically, being associated wi'~h a, decrease in the blood HDL level).
While the present invention has bean described specifically in detail with reference f.4 certa~.n embodiments and examples thereof, it would be apparent that i.t is passible to practice it in other xoxms. 2n light of the disclosure, i.t wi,l,l be understood that various modifications and variations are within the spirit and scope of the appended claims.
~5

Claims (5)

1. An ABCA1 stabilizer comprising an effective amount of a bisphenol-type compound selected from the group consisting of probucol spiroquinone, probucol diphenoquinone, probucol bisphenol, and salts thereof.

2. A prophylactic/therapeutic agent for low-HDL
cholesterolemia, comprising at least one ABCA1 stabilizer according to claim 1.

3. A prophylactic/therapeutic agent for arteriosclerosis, comprising at least one ABCA1 stabilizer according to claim 1.

4. A drug comprising a member selected from the group consisting of ABCA1 stabilizers, prophylactic/therapeutic agents for low-HDL cholesterolemia, and prophylactic/therapeutic agents for arteriosclerosis, according to any of claims 1 to 3, in combination with at least one drug selected from the group consisting of antidiabetes drugs, therapeutic drugs for complications of diabetes, antiobesity drugs, antihypertensive drugs, antihyperlipidemic drugs, diuretics, antithrombotic drugs, and anti-Alzheimer drugs.

5. An agent for increasing a blood level of HDL, comprising an effective amount of a bisphenol-type compound selected from the group consisting of probucol spiroquinone, probucol diphenoquinone, probucol bisphenol, and salts thereof.

To provide a pharmaceutically effective prophylactic/preventive agent for low-HDL cholesterolemia, focusing on an HDL-generating mechanism. The ABCA1 stabilizer of the present invention contains a bisphenol-type compound selected form probucol spiroquinone, probucol diphenoquinone, and probucol bisphenol as an effective ingredient. The ABCA1 stabilizer can continuously and stably express ABCA1 by a mechanism quite different from that of conventional processes, and thus is useful as prophylactic/preventive agent for low-HDL cholesterolemia or arteriosclerosis.